Hi everyone, I have an interesting situation which involved samples with degraded RNA. I would like to get some comments on it, and a search of the archives indicated that an example of what degraded RNA looks like on an Affy chip would be useful for others. The samples are from E. coli, which has notoriously unstable RNA with half-lifes on the order of minutes. When grown in anaerobic conditions, the RNA becomes even more unstable. Many of the total RNA samples that our core facility was receiving from the researchers were degraded, even though they were supposedly fine after extraction. Due to a variety of reasons, our core decided to label and hyb a sample that was completely degraded according to a Bioanalyzer. We eventually were able to get non- degraded total RNA for all of the samples. When I compare the degraded sample to the other samples, it is indeed an outlier, but it has HIGHER pm and mm signals than the non-degraded samples. The density plots show an interesting bimodal distribution for all the samples, and a disturbing trend towards the shape of the degraded sample. I say disturbing because the samples grown in anaerobic conditions are closer to the degraded sample than the samples grown in aerobic conditions. The pm, mm and both density plots can be seen here (the degraded sample is the green line with the largest right-hand peak): ftp://ftp.biotec.uiuc.edu/pub/Ecoli_figures/ Oddly, when I look at the RNA digestion plot (which may or may not show degradation, according to what I found in the archives), all of the samples included the degraded sample have flat slopes; only a couple had p<0.05, and they had slightly negative slops; see above link for plot (degraded sample in green, anaerobic samples in red; plotting was done with transform="neither"). What also surprises me is the huge difference between gc-based background correction of GCRMA and either of the background corrections used by RMA or MAS5 (I've traced it to the background correction). When the gc-based background correction is used then median polish to summarize the values (no normalization), the degraded sample has, as expected, almost no signal, but using either RMA or MAS5 (without normalization) results in the degraded sample having the HIGHEST signals. And the closer a sample's raw distribution was to the degraded sample, the more it followed the same pattern for background correction (see the above link for boxplots; the degraded sample is the last one on the right, #423; even numbers are aerobic samples, odd are anaerobic samples). Based on the behavior of the degraded sample, I would say that the gc- based background correction is the one to use. It also appears that all of the aerobic samples, but only 3 of the anaerobic samples (417, 419, 423B) have relatively little degradation, and the rest of the anaerobic samples are severely degraded. Re-doing these samples is unfortunately NOT an option at this point. The researchers want me to go ahead with the statistical analysis, even though I have told them that any changes will be primarily driven by degradation, and not real expression levels. I probably should not use any normalization method because the assumption of few changes is not met. Would you agree with my conclusions above, or do you have alternative interpretations and suggestions? Is it possible that prokaryotic RNA and its N-terminus labeling method are different enough from eukaryotic RNA and the biotin-labeled nucleotide labeling method so that these functions (particularly gc-based bg correction) should not be used? Thanks in advance, Jenny Jenny Drnevich, Ph.D. Functional Genomics Bioinformatics Specialist W.M. Keck Center for Comparative and Functional Genomics Roy J. Carver Biotechnology Center University of Illinois, Urbana-Champaign 330 ERML 1201 W. Gregory Dr. Urbana, IL 61801 USA ph: 217-244-7355 fax: 217-265-5066 e-mail: drnevich at uiuc.edu

I should be more precise about the prokaryotic labeling method... Fragmented cDNA is labeled at the 3' end, then hybridized to antisense arrays. Affy's manual only says the labeling is done with GeneChip DNA Labeling Reagent, but the original reference used 70 ?M biotin-N6-ddATP (http://www.pubmedcentral.gov/articlerender.fcgi?tool=pubmed&pubmedid= 11713332] At 01:43 PM 2/28/2006, Jenny Drnevich wrote: > Is it possible that prokaryotic RNA and >its N-terminus labeling method are different enough from eukaryotic RNA and >the biotin-labeled nucleotide labeling method so that these functions >(particularly gc-based bg correction) should not be used? > >Thanks in advance, >Jenny > > >Jenny Drnevich, Ph.D. > >Functional Genomics Bioinformatics Specialist >W.M. Keck Center for Comparative and Functional Genomics >Roy J. Carver Biotechnology Center >University of Illinois, Urbana-Champaign > >330 ERML >1201 W. Gregory Dr. >Urbana, IL 61801 >USA > >ph: 217-244-7355 >fax: 217-265-5066 >e-mail: drnevich at uiuc.edu > >_______________________________________________ >Bioconductor mailing list >Bioconductor at stat.math.ethz.ch >https://stat.ethz.ch/mailman/listinfo/bioconductor